Genetic Disruption of Kir6.2, the Pore-Forming Subunit of ATP

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Genetic Disruption of Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K؉ Channel, Predisposes to Catecholamine-Induced Ventricular Dysrhythmia Xiao-Ke Liu,1 Satsuki Yamada,1 Garvan C. Kane,1 Alexey E. Alekseev,1 Denice M. Hodgson,1 Fearghas O’Cochlain,1 Arshad Jahangir,1 Takashi Miki,2 Susumu Seino,2 and Andre Terzic1

-؉ cellular well-being and stress adaptation (9–11). Specifi Metabolic-sensing ATP-sensitive K channels (KATP channels) adjust membrane excitability to match cellu- cally, under catecholamine surge, proper KATP channel lar energetic demand. In the heart, KATP channel activ- activity is required for the coordinated adjustment of ity has been linked to homeostatic shortening of the membrane-dependent cellular functions, including ade- action potential under stress, yet the requirement of quate calcium handling and sustained contractility (9,10). channel function in securing cardiac electrical stability Although KATP channel opening has been associated with is only partially understood. Here, upon catecholamine homeostatic shortening of the cardiac action potential challenge, disruption of KATP channels, by genetic dele- under increased metabolic demand (6,9), the precise role tion of the pore-forming Kir6.2 subunit, produced defec- of channel function in support of cardiac electrical stabil- tive cardiac action potential shortening, predisposing the myocardium to early afterdepolarizations. This def- ity is only partially understood. Thus, this study was icit in repolarization reserve, demonstrated in Kir6.2- designed to address the contribution of KATP channels in knockout hearts, translated into a high risk for membrane electrical tolerance in the heart under adrener- induction of triggered activity and ventricular dys- gic stress. rhythmia. Thus, intact KATP channel function is manda- To this end, the electrical consequences of adrenergic tory for adequate repolarization under sympathetic stress were tested in hearts lacking the pore-forming stress providing electrical tolerance against triggered subunit of K channels, through genetic disruption of arrhythmia. Diabetes 53 (Suppl. 3):S165–S168, 2004 ATP Kir6.2, and compared with the wild type. In KATP channel knockout hearts, sympathomimetic challenge unmasked an inadequate repolarization reserve predisposing to abnormal action potentials with afterdepolarizations and xpressed at high density in the cardiac sarco- ϩ inducing ventricular dysrhythmia. Hence, KATP channels lemma, ATP-sensitive K (KATP) channels are heteromultimers of the pore-forming Kir6.2 sub- are required for electrical adaptation that protects against unit with the regulatory sulfonylurea receptor triggered arrhythmia within the adrenergically stressed E myocardium. (1). By virtue of a unique ability to decode signals of cellular energetic distress, KATP channels adjust membrane electrical activity in response to metabolic demand RESEARCH DESIGN AND METHODS (2,3). Disturbances in KATP channel function, either Kir6.2-knockout mice. Mice deficient in KATP channels were generated by through pharmacological blockade with sulfonylurea med- targeted disruption of the KCNJ11 gene, which encodes the pore-forming Kir6.2 subunit of the channel complex (12). Kir6.2-knockout mice were ication or through genetic mutation of channel proteins, backcrossed for five generations into a C57BL/6 background. This investiga- have been linked to increased susceptibility for develop- tion was approved by the Mayo Clinic Institutional Animal Care and Use ment and progression of cardiovascular conditions (4–8). Committee. In particular, in hyperadrenergic states, ranging from In situ aortic cannulation and Langendorff perfusion. Mice were anes- physical exertion to decompensated heart failure, cardiac thetized with intraperitoneal injection of 2,2,2-tribromoethanol (0.375 mg/g body wt; Sigma), intubated, and ventilated, and the aortic root was cannulated KATP channels have been implicated in the maintenance of in situ (10). Perfusion was sustained ex vivo on a Langendorff system, at 90 cm

H2O with 37°C-prewarmed and 100% O2-bubbled Tyrode solution (in mmol/l: NaCl 137, KCl 5.4, CaCl 2, MgCl 1, HEPES 10, and glucose 10, pH 7.4 with 1 2 2 From the Division of Cardiovascular Diseases, Department of Medicine, NaOH). After a 10-min equilibration, KCl was reduced to 2.7 mmol/l and MgCl Department of Molecular Pharmacology and Experimental Therapeutics, 2 to 0.5 mmol/l, with the atrioventricular node cauterized to allow ventricular Mayo Clinic College of Medicine, Rochester, Minnesota; and the 2Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medi- pacing (13). Coronary flow was monitored with a T106 blood flow meter cine, Kobe, Japan. (Transonic Systems). Address correspondence and reprint requests to Andre Terzic, MD, PhD, Electrogram and monophasic action potential recordings. Orthogonal Guggenheim 7, Mayo Clinic, Rochester, MN 55905. E-mail: terzic.andre electrogram signals were simultaneously recorded using four silver-silver @mayo.edu. chloride electrodes surrounding the perfused heart in a simulated “Einthoven” Received for publication 12 March 2004 and accepted in revised form 18 configuration, and signals were amplified by an electrocardiographic amplifier May 2004. (Gould Electronics). A catheter (NuMed) was placed in the left ventricular This article is based on a presentation at a symposium. The symposium and endocardium to pace the heart at twice diastolic threshold intensity with 2-ms the publication of this article were made possible by an unrestricted educa- tional grant from Servier. pulse width and 100-ms cycle length using a pulse generator (A310 Accu- pulser; World Precision Instruments). Monophasic action potentials were APD90, action potential duration at 90% repolarization; KATP channel, ATP-sensitive Kϩ channel; Kir6.2-KO, Kir6.2 knockout. continuously recorded from the left ventricle by a probe (EP Technologies) © 2004 by the American Diabetes Association. positioned on the epicardial surface, and amplified signals (IsoDam; World

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S165 KATP CHANNELS AND TRIGGERED ARRHYTHMIA

FIG. 1. Isoproterenol challenge induced

action potential shortening (APD90)in wild-type (WT) (A) but not Kir6.2-KO (B) hearts, which developed early afterdepolarizations (EAD) (C). D: Inci- dence of EAD in the initial 50 action potentials after 5 min of isoproterenol infusion.

Precision Instruments) were acquired at 11.8 kHz and stored for off-line digital After a 10-min perfusion with the sympathomimetic iso- analysis (9). proterenol (1 ␮mol/l), monophasic action potential dura- Whole-cell patch clamp recording from isolated cardiomyocytes. Cardi- Ϯ omyocytes were enzymatically dissociated from the ventricular myocardium tion at 90% repolarization (APD90) shortened from 82 2 (10). Action potentials were recorded at 30 Ϯ 1°C from current-clamped to 74 Ϯ 2 ms in wild-type hearts (P Ͻ 0.01, n ϭ 6; Fig. 1A). isolated cells paced at 1 Hz, and were superfused with Tyrode solution (pH 7.2 Ϯ Ϯ In contrast, APD90 remained at 79 3 and 80 3ms adjusted with KOH) using the whole-cell patch clamp technique with 5–10 before and following isoproterenol treatment, respec- mol/l⍀ pipettes containing (in mmol/l) KCl 120, MgCl 1, Na ATP 5, HEPES 10, 2 2 tively, in Kir6.2-KO hearts (n ϭ 6) (Fig. 1B). This deficit in EGTA 0.5, and CaCl2 0.01 (14). Statistics. Comparisons were made using the Student’s t test. A significance repolarization led to distorted action potential profiles level of 0.05 was preselected. Data are reported as means Ϯ SE. with characteristic phase 3 early afterdepolarizations man- ifested as distinct humps in hearts lacking functional KATP RESULTS AND DISCUSSION channels (Fig. 1B and C). In all Kir6.2-KO hearts (n ϭ 8), Whereas at baseline the action potentials were similar, the adrenergic challenge induced early afterdepolarizations, metabolic challenge of adrenergic stimulation induced which occurred in 97 Ϯ 2% of the action potentials distinct outcomes depending on the presence of functional examined (Fig. 1D). This is in contrast to the action ϭ KATP channels, with significant shortening of the action potential profile of the wild type (n 6) that maintained a potential duration observed in wild-type hearts but not in smooth repolarization contour following isoproterenol age- and sex-matched counterparts lacking the Kir6.2 challenge (Fig. 1A) without significant afterdepolariza- pore-forming channel subunit (Kir6.2-KO) (Fig. 1A and B). tions (1 Ϯ 1%; P Ͻ 0.01 vs. Kir6.2-KO) (Fig. 1D).

S166 DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 X.-K. LIU AND ASSOCIATES

FIG. 2. A: Similar coronary ﬂow in the absence and presence of isoproterenol (1 ␮mol/l) in wild-type (WT) and Kir6.2-KO hearts. B: Isoproterenol-induced abnormal repolarization with early afterdepolarization in an isolated current-clamped Kir6.2-KO cardi- omyocyte.

Abnormal electrical response of Kir6.2-KO hearts under ogous to hearts with genetic and/or environmental adrenergic challenge was not associated with an isoprot- compromise of repolarizing currents, as observed in con- erenol-induced deficit in coronary perfusion (Fig. 2A). In genital or acquired long QT syndrome (17), here isopro- fact, abnormal electrical activity during repolarization terenol challenge was proarrhythmic in the KATP channel– observed at the whole-heart level was reproduced at the deficient myocardium provoking afterdepolarizations and single-cell level using action potential recording in isopro- triggered activity. This is in line with pharmacological terenol-stressed current-clamped Kir6.2-KO cardiomyo- studies that demonstrate that KATP channel activation with cytes (Fig. 2B). potassium channel openers prevents—whereas channel Afterdepolarizations in isoproterenol-challenged Kir6.2- blockade with sulfonylurea drugs enhances—afterdepolar- KO hearts translated into increased electrical vulnerability izations and triggered activity (18–20). In fact, in a recent (Fig. 3). In the absence of functional KATP channels, randomized clinical trial in patients with type 2 diabetes, afterdepolarizations induced triggered activity, disrupting the sulfonylurea glyburide, but not the alternative oral regular rhythm and manifesting as premature ventricular hypoglycemic agent metformin, caused an increase in QT complexes on the electrogram (Fig. 3A). On average, prolongation with QT dispersion on the electrocardiogram isoproterenol-induced afterdepolarizations complicated (21). Moreover, mutations in the cardiac sulfonylurea by triggered activity were observed in six of eight receptor inducing deficits in KATP channel function have Kir6.2-KO (75%) compared with one of six wild-type (16%) been identified in patients with cardiomyopathy and ven- hearts, translating into a 16-fold higher risk (P Ͻ 0.05) of tricular arrhythmia (8). developing premature ventricular complexes (Fig. 3B). Suppression of KATP channel activity, whether by ge- Hence, absence of KATP channels produces a deficit in the netic deletion of channel subunits or through use of repolarization reserve leading to a pronounced suscepti- channel antagonists, predisposes to inadequate calcium bility of Kir6.2-KO hearts to isoproterenol-induced ventric- handling and intracellular calcium overload (5,9). In turn, ular dysrhythmia. Thus, sarcolemmal KATP channels excessive cytosolic calcium acts as a trigger for early provide for membrane electrical stability that reduces the depolarizations (22,23). Conversely, in the intact heart, ␤ risk for arrhythmia under hyperadrenergic conditions. where KATP channel opening is promoted by -adrenergic– Imposed catecholamine stress is a well-established pre- mediated phosphorylation of channel proteins (24,25) or cipitator of triggered activity and arrhythmia (15,16). Anal- subsarcolemmal ATP depletion (26), shortening of cardiac

FIG. 3. A: Kir6.2-KO hearts with early afterdepolarizations (EAD) demonstrated triggered activity on monophasic action potentials (MAP) and premature ventricular complexes (PVC) on electro- grams (EG). For comparison, an EG is shown from wild-type hearts that are not prone to EAD-triggered activity/PVC. B: Incidence of triggered activity with ac- companying PVC in the initial 50 action potentials following 5 min of isoproterenol infusion.

DIABETES, VOL. 53, SUPPLEMENT 3, DECEMBER 2004 S167 KATP CHANNELS AND TRIGGERED ARRHYTHMIA action potentials would balance catecholamine-induced 9. Zingman LV, Hodgson DM, Bast PH, Kane GC, Perez-Terzic C, Gumina RJ, increase in calcium influx protecting against triggered Pucar D, Bienengraeber M, Dzeja PP, Miki T, Seino S, Alekseev AE, Terzic A: Kir6.2 is required for adaptation to stress. Proc Natl Acad SciUSA arrhythmia. 99:13278–13283, 2002 In summary, the present study underscores the homeo- 10. Hodgson DM, Zingman LV, Kane GC, Perez-Terzic C, Bienengraeber M, static requirement for functional KATP channels in the Ozcan C, Gumina RJ, Pucar D, O’Coclain F, Mann DL, Alekseev AE, Terzic adaptation of cardiac repolarization under adrenergic A: Cellular remodeling in heart failure disrupts KATP channel-dependent stress. In this regard, a deficit in K channel function is stress tolerance. EMBO J 22:1732–1742, 2003 ATP 11. 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